Lyme disease is a complex multisystem disorder caused by the tick-borne spirochete Borrelia buradorferi. This disease has three clinical stages that can overlap or occur alone: stage one--early disease, including a characteristic expanding skin lesion (erythema chronicum migrans) and constitutional flu-like symptoms; stage two--cardiac and neurological disease; and stage three--arthritis and chronic neurological syndromes.
Presently, the incidence of reported Lyme disease is increasing, which is probably due to improved awareness and recognition of the disease, as well as to an actual increase in incidence and geographic spread. B. burgdorferi can be isolated from blood or skin biopsies taken from acutely ill patients, but the yield is low and the procedures are difficult. Serologic testing for antibodies using an enzyme-linked immunosorbent assay (ELISA) or indirect immunofluorescence assay (IFA) is the standard method used to confirm a clinical diagnosis, but current tests are poorly standardized, and false-negative or false-positive results can occur (Barbour, (1989) Ann Intern Med 110:501). In addition to misdiagnosis caused by lack of standardization of serologic testing, cross-reactivity with Treponema and with other Borrelia may occur. Patients with stage one or two disease may be seronegative because it may take as long as three to six months after exposure for antibodies to become detectable with currently available tests. Patients who develop later stages of the illness may occasionally be seronegative if they were treated acutely with antibiotics (Dattwyler et al., (1988) N Engl J Med 319:1441). Previously untreated patients with a late stage of the disease are, apparently, almost always seropositive.
The use of specific polynucleotide sequences as probes for the recognition of infectious agents is becoming a valuable alternative to problematic immunological identification assays. For example, PCT publication W084/02721, published Jul. 19, 1984 describes the use of nucleic acid probes complementary to targeted nucleic acid sequences composed of ribosomal RNA, transfer RNA, or other RNA in hybridization procedures to detect the target nucleic acid sequence. While the assay may provide greater sensitivity and specificity than known DNA hybridization assays, hybridization procedures which require the use of a complementary probe are generally dependent upon the cultivation of a test organism and are, therefore, unsuitable for rapid diagnosis.
Polymerase chain reaction (PCR) is a powerful technique that can be used for the detection of small numbers of pathogens whose in vitro cultivation is difficult or lengthy, or as a substitute for other methods which require the presence of living specimens for detection. In its simplest form, PCR is an in vitro method for the enzymatic synthesis of specific DNA sequences, using two oligonucleotide primers that hybridize to opposite strands and flank the region of interest in the target DNA. A repetitive series of cycles involving template denaturation, primer annealing, and the extension of the annealed primers by DNA polymerase results in the exponential accumulation of a specific fragment whose termini are defined by the 5' ends of the primers. PCR reportedly is capable of producing a selective enrichment of a specific DNA sequence by a factor of 10.sup.9. The PCR method is described in Saiki et al., (1985) Science 230:1350 and is the subject of U.S. Pat. Nos. 4,683,195; 4,683,202; 4,800,159; and 4,965,188. This method has been used to detect the presence of the aberrant sequence in the beta-globin gene which is related to sickle cell anemia (Saiki et al., (1985) supra) and the human immunodeficiency virus (HIV) RNA (Byrne et al., (1988) Nuc Acids Res 16:4165). However, before the method can be used, enough of the nucleotide sequence of the disease-associated polynucleotide must be known to design primers for the amplification, and to design probes specific enough to detect the amplified product.
Both genomic and plasmid DNA sequences have been used for the identification of Borrelia, but neither of these methods have been shown to detect all Borrelia burgdorferi isolates. Schwan et al., (1989) J Clin Microbiol 27:1734 and (1988) Ann NY Acad Sci 539:419! use nucleic acid hybridization probes derived from the 49 kilobase linear plasmid of B. burgdorferi for the detection and identification of B. burgdorferi from a number of other Borrelia species in the United States. A nucleic acid probe containing the 5' portion of the variable major protein 7 (vmp 7) of B. hermsii (the causative agent of tick-borne relapsing fever) was also employed in these studies.
One of the inherent limitations of this assay is its dependency on the identification of plasmid DNA. Such identification procedures are inherently unreliable given that plasmids, over time, are unstable in cultured spirochetes or may be absent from some natural isolates of the pathogen or clinical specimens.
Rosa and Schwan (1989) J Infect Dis 160(6):1018 used PCR to amplify a target selected from randomly cloned B. burgdorferi DNA. While the specificity of the PCR assay suggested a utility for this technique, not all B. burgdorferi isolates were detected. Since it is not known currently whether Lyme disease is caused by all isolates of B. burgdorferi, it is essential for accurate diagnosis to demonstrate that all isolates will be detected in any given assay system. Furthermore, their system is not sensitive enough to detect the pathogen in clinical or biological specimens.
In light of current limitations for the serological identification of Lyme disease, it would be desirable to provide a rapid and sensitive procedure for the detection of B. burgdorferi in a clinical sample suspected of containing the spirochete. It would also be desirable to develop reagents that are useful for detecting all geographical isolates of B. burgdorferi.